The objective of this project is to explore how geometric cues in the niche guide mesenchymal stem cells (MSCs) towards specific fates. Human MSCs are multipotent stem cells that differentiate into fat, bone, tendon, muscle and neuron-like cells. The multipotency and ease of collection (i.e. fat aspirates, bone marrow) makes MSCs one of the more widely studied stem cell types for regenerative therapy and tissue engineering. Significant strides have been made in determining the chemical and biological signals that direct MSC fate decisions. However, there is currently very little understanding of the role of the niche geometry in directing MSC self-renewal and lineage commitment. The majority of systems that attempt to model the microenvironment of stem cells in their niche do not adequately control for variations in cell shape, spatial distribution and proliferation. The proposed research aims to use model surfaces to study the differentiation of an array of single MSCs confined to precise shapes. Cell adhesion and cytoskeletal features are important in many aspects of tissue morphogenesis. Thus, the choice of shapes for this work will be influenced by specific geometric cues previously shown to direct adhesive, contractile and protrusive elements. As the first specific aim, MSCs will be cultured on mixed patterns of shapes under growth and differentiation conditions to evaluate the effect of geometric cues by themselves and in combination with lineage promoting soluble factors. Histochemistry, immunofluorescence and RT-PCR will be used to compare cells across conditions and shapes. The second specific aim will explore inhibition of cytoskeletal elements and lineage regulators to isolate important biochemical pathways in shape-promoted fate decisions. Furthermore, the role of focal adhesion density within the niche will be explored in parallel by varying the surface chemistry across shapes. Along with techniques developed in aim 1, DNA microarray analysis, protein electrophoresis, western blotting and enzyme activity assays will be used to isolate important networks towards a thorough understanding of signaling events within the MSC niche. Mesenchymal stem cells (MSCs) from adult bone marrow hold promise as an abundant source of multipotent cells for regenerative medicine. Improper MSC fate regulation has been implicated in numerous pathologies including: osteoporosis, atherosclerosis and cancer cell metastasis. Therefore, deciphering how an adherent stem cells shape influences its fate is of vast importance for both understanding regenerative and pathological processes as well as for designing materials for stem cell based therapies.